基于网络药理学和实验验证探讨玉泉胶囊治疗糖尿病肾病的作用机制

Mechanism of Yuquan Capsule in Treating Diabetic Nephropathy Based on Network Pharmacology and Experimental Valida⁃tion

[目的]运用网络药理学和体外实验探讨玉泉胶囊治疗糖尿病肾病的潜在作用机制。[方法]借助中药系统药理学数据库与分析平台(Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform,TCMSP)和中医药综合数据库(Traditional Chinese Medicine Integrated Database,TCMID)筛选玉泉胶囊有效成分及靶点基因,基因组注释数据平台(Genome Annotation Database Platform,GeneCards)、疾病基因网络(Disease Gene Network,DisGeNET)数据库和治疗靶点数据库(Therapeutic Target Database,TTD)筛选糖尿病肾病的疾病相关基因,STRING 11.5数据库构建蛋白互作(protein-protein interaction,PPI)网络,并利用Cytoscape 3.7.2软件可视化,利用Cytoscape 3.7.2软件的CytoHubba和MCODE插件筛选关键化合物与核心靶点,利用R软件(ClusterProfiler包)进行基因本体(gene ontology,GO)富集分析和京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)信号通路富集分析,利用AutoDock和PyMOL软件进行分子对接验证,最后对网络药理学预测结果进行体外实验验证。[结果]玉泉胶囊抗糖尿病肾病活性成分有177个,活性成分作用靶点共477个,作用于糖尿病肾病的靶点有135个,核心靶点包括丝氨酸/苏氨酸激酶1(serine/threonine kinase 1,AKT1)、白细胞介素-1β(interleukin-1 beta,IL1B)、过氧化氢酶(catalase,CAT)、一氧化氮合酶(nitric oxide synthase 3,NOS3)、瘦素(leptin,LEP)、胰岛素样生长因子1(insulin-like growth factor 1,IGF1)和趋化因子配体2(C-C motif chemokine ligand 2,CCL2);GO富集分析共得到相关条目2557个,其中生物过程1415个、细胞组成657个、分子功能48个;KEGG通路分析结果显示玉泉胶囊作用于糖尿病肾病的靶点富集在66条通路上,其中环磷酸腺苷(cyclic adenosine monophosphate,cAMP)信号通路、高级糖基化终末产物-受体(advanced glycation end products-receptor for advanced glycation end products,AGE-RAGE)信号通路在糖尿病并发症中的作用等可能是玉泉胶囊治疗糖尿病肾病的关键通路。分子对接结果显示核心靶点与关键化合物均能较好地结合。实验验证显示,CCL2蛋白水平呈药物剂量依赖性下降,CAT、内皮型一氧化氮合酶(endothelial nitric oxide synthase,eNOs)蛋白水平呈药物剂量依赖性上升。[结论]玉泉胶囊治疗糖尿病肾病具有多成分、多靶点的特点,其机制可能与下调CCL2、上调CAT、eNOs的蛋白表达,抑制糖尿病肾病的氧化应激,调节关键通路相关。

[Objective]To investigate the potential mechanism of Yuquan Capsule in treating diabetic nephropathy(DN)by network pharmacology and in vitro experiments.[Methods]The active ingredients and disease-associated genes of Yuquan Capsule were screened by Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP)and Traditional Chinese Medicine Integrated Database(TCMID),the target genes of DN were screened by Genome Annotation Database Platform(GeneCards),Disease Gene Network(DisGeNET)database and Therapeutic Target Database(TTD).The protein-protein interaction(PPI)network was drawn by STRING 11.5 database,and visualized by Cytoscape 3.7.2 software.The key compounds and core targets were screened by CytoHubba and MCODE plugins of Cytoscape 3.7.2 software.Gene ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis were performed by R software(ClusterProfiler package),and the molecular docking validation was performed by AutoDock and PyMOL software.[Results]There were 177 Yuquan Capsule against DN,477 active components and 135 targets of Yuquan Capsule against DN.The core targets included serine/threonine kinase 1(AKT1),interleukin-1 beta(IL1B),catalase(CAT),nitric oxide synthase 3(NOS3),leptin(LEP),insulin-like growth factor 1(IGF1)and C-C motif chemokine ligand 2(CCL2).GO enrichment analysis showed 2557 related items,including 1415 biological processes,657 cellular components and 48 molecular functions.KEGG pathway analysis showed that the targets of Yuquan Capsule against DN were enriched in 66 pathways,of which cyclic adenosine monophosphate(cAMP)signaling pathway and advanced glycation end products-receptor for advanced glycation end products(AGE-RAGE)signaling pathway in diabetic complications may be the key pathways of Yuquan Capsule in the treatment of DN.Molecular docking results showed that the core target and key compounds could bind well.Experimental verification showed that the protein level of CCL2 decreased in a drugdose-dependent manner,and the protein level of CAT and endothelial nitric oxide synthase(eNOs)increased in a drugdose-dependent manner.[Conclusion]Yuquan Capsule has the characteristics of multiple components and multiple targets in the treatment of DN.The mechanism may be related to the down-regulation of CCL2 and up-regulation of CAT and eNOs protein expression,inhibiting oxidative stress,and regulating the key pathway.